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Morphogenesis of the Node of Ranvier: Co-Clusters ofAnkyrin and Ankyrin-Binding Integral Proteins Define EarlyDevelopmental Intermediates

Stephen Lambert, Jonathan Q. Davis, and Vann Bennett

Department of Cell Biology and the Howard Hughes Medical Institute, Duke University Medical Center, Durham,North Carolina 27710

AnkyrinG 480/270 kDa and three ankyrin-binding integral mem-brane proteins (neurofascin, NrCAM, and the voltage-dependent sodium channel) colocalize within a specialized do-main of the spectrin–actin network found at axonal segments ofnodes of Ranvier in myelinated axons. Before myelination inembryonic nerves, ankyrinG 480/270 kDa and the relatedankyrin isoform ankyrinB 440 kDa are co-expressed along withNrCAM in an abundant, continuous distribution along thelength of axons. This study has resolved intermediate stages inthe developmental transition from a continuous distribution ofankyrinG 480/270 kDa in all axons to a highly polarized local-ization at the node of Ranvier in the developing rat sciatic nerve.The first detected event is formation of clusters containing thecell adhesion molecules neurofascin and NrCAM at sites inde-pendent of myelin-associated glycoprotein (MAG)-stainingSchwann cell processes. Subsequent steps involve recruitmentof ankyrinG 480/270 kDa and the voltage-dependent sodium

channel to cluster sites containing cell adhesion molecules, andelaboration of MAG-staining Schwann cell processes adjacentto these cluster sites. Formation of the mature node of Ranvierresults from the fusion of asynchronously formed pairs of clus-ters associated with MAG-positive Schwann cells flanking thesite of presumed node formation. Studies with the hypomyeli-nating mutant mouse trembler demonstrate that the elaborationof compact myelin is not required for the formation of theseclustered nodal intermediates. Clustering of neurofascin andNrCAM precedes redistribution of ankyrinG 480/270 kDa andthe voltage-dependent sodium channel, suggesting that theadhesion molecules define the initial site for subsequent as-sembly of ankyrin and the voltage-dependent sodium channel.

Key words: node of Ranvier; cell adhesion molecules; ankyrin;voltage-dependent sodium channel; myelination; tremblermutant

The localization of ion channels, in particular the voltage-dependent sodium channel, to gaps in the myelin sheath known asthe nodes of Ranvier, is crucial to the propagation of saltatoryaction potentials in myelinated nerves. In the internodal axonalmembrane, the voltage-dependent sodium channel is found atconcentrations of 20–25 channels/mm2, compared with concen-trations ranging from 1000 channels/mm2 (Shrager, 1989) to12,000 channels/mm2 (Ritchie and Rogart, 1977) at the node ofRanvier. The asymmetric distribution of ion channels in themyelinated axon and the regular spacing of these nodes at ;100times the axonal diameter (Rushton, 1951; Chiu, 1980) are criticalfor the fast conduction velocities associated with these axons andrepresent an interesting problem in cell polarity and membranedifferentiation.

A clue as to the molecular organization of the node comes fromobservations that the voltage-dependent sodium channel co-purifies and binds with high affinity to the peripheral membraneprotein ankyrin (Srinivasan et al., 1988). In addition, otherankyrin-binding proteins are also localized at the node, includingisoforms of neurofascin and NrCAM (Davis et al., 1993, 1996),

ankyrin-binding cell adhesion molecules related to L1 (for re-view, see Hortsch, 1996). Ankyrin itself is localized at the 1- to2-mm-size nodal axonal plasma membrane (Kordeli et al., 1990)and is a component of the electron dense undercoating observedbeneath the nodal membrane (Peters, 1966; Ichimura and Ellis-man, 1991). Ankyrins are a family of spectrin-binding proteinsthat associate with diverse integral proteins, including ion chan-nels, calcium-release channels, and cell adhesion molecules, andlink these proteins to the spectrin-based membrane skeleton (forreview, see Bennett and Gilligan, 1993; Lambert and Bennett,1993). The isoforms of ankyrin localized at nodes of Ranvierhave recently been identified as 480 and 270 kDa alternativelyspliced variants of the ankyrinG gene (Kordeli et al., 1995). Theseankyrin isoforms are concentrated at axon initial segments andnodes of Ranvier in the adult rat CNS and PNS (Kordeli et al.,1995), and to our knowledge they represent the first uniquecytoplasmic components of the node to be identified. Nodalankyrin isoforms are distinguished by a 46 kDa serine–threonine-rich domain glycosylated with O-GlucNAc residues(Zhang and Bennett, 1996), and like other ankyrins associatewith integral proteins through a membrane-binding domain com-prising 24 ANK repeats. Biochemical studies of this domain(Michaely and Bennett, 1995a,b) suggest that ankyrin could fa-cilitate lateral homo- and heterocomplexes between integralmembrane proteins.

The absence of suitable molecular markers has hindered elu-cidation of the role of components of the electron-dense under-coating in the differentiation of the nodal membrane. This study

Received March 20, 1997; revised May 29, 1997; accepted July 9, 1997.This work was supported by the Howard Hughes Medical Institute and a grant

from National Institutes of Health (DK29808). Zhang Xu is gratefully acknowl-edged for the preparation of the chicken anti-ankyrinG common “tail” antibody.

Correspondence should be addressed to Dr. Lambert at his present address:Worcester Foundation for Biomedical Research, 222 Maple Avenue, Shrewsbury,MA 01545.Copyright © 1997 Society for Neuroscience 0270-6474/97/177025-12$05.00/0

The Journal of Neuroscience, September 15, 1997, 17(18):7025–7036

examines the distribution of ankyrinG 480/270 kDa, the voltage-dependent sodium channel, neurofascin, and NrCAM duringdevelopment in the myelinating sciatic nerve. AnkyrinG 480/270kDa polypeptides are highly expressed in a uniform distributionalong premyelinated axons at embryonic day 16 and redistributeto clusters adjacent to the ends of Schwann cells in early postnataldevelopment. The voltage-dependent sodium channel also is lo-calized in clusters, as reported recently in developing and regen-erating nerves (Dugandzija-Novakovic et al., 1995; Vabnick et al.,1996), and is colocalized at these sites with ankyrinG 480/270 kDaas well as neurofascin and NrCAM. Clustering of neurofascin andNrCAM precedes redistribution of ankyrinG 480/270 kDa andthe voltage-dependent sodium channel and also precedes earlyevents in myelin formation, as determined by the expression ofmyelin-associated glycoprotein (MAG). These results and thefinding of clusters in hypomyelinating mice suggest that axonaladhesion molecules define the initial site for subsequent assemblyof ankyrin and the voltage-dependent sodium channel into thedifferentiating nodal axonal membrane and that these events areindependent of the formation of compact myelin.

MATERIALS AND METHODSPreparation of antibodies. Antibodies against the common tail region ofrat ankyrinG 480 and 270 kDa were raised in chickens for double-labelingstudies. Chickens were immunized with a purified recombinant polypep-tide corresponding to the rat equivalent of residues 1821–2337 of thehuman ankyrinG sequence. This polypeptide was produced by expressionin bacteria of the rat cDNA using the pGemex expression vector (Pro-mega, Madison, WI), resulting in a fusion product with the viral gene 10protein. Antibodies were initially purified from chicken egg yolk using anEgg Yolk Purification Kit (Pharmacia Biotech, Piscataway, NJ) andaffinity-purified against purified recombinant polypeptide immobilizedon Sepharose CL-6B (Pharmacia) after the previous depletion of anti-bodies using immobilized gene 10 polypeptide. Antibodies against neu-rofascin and NrCAM were generated in rabbits using purified recombi-nant FNIII domains (residues 581–1020 of rat neurofascin and residues583–1018 of rat NrCAM) from these molecules as antigens. Theserecombinant domains were prepared in bacteria as above. Affinity-purified antibodies were then prepared from sera by purification againstimmobilized native 186 kDa neurofascin and NrCAM. Native proteinswere purified from detergent extracts of adult rat brain membranes asdescribed previously (Davis et al., 1993). Antibodies against neurofascindid not cross-react with purified native NrCAM by immunoblot analysisand vice versa (Davis et al., 1996). Antibodies against the voltage-dependent sodium channel were prepared by immunization of rabbitswith four multiple-antigen peptides corresponding to residues 440–453,482–492, 547–561, and 582–600 of the aI subunit (Noda et al., 1986).These sequences are relatively conserved in the three a subunits ex-pressed in the rat brain and are in an area of the molecule proposed tobe a cytoplasmic linker between the I and II transmembrane pore-forming units. An area of the rat aI subunit (corresponding to nucleo-tides 1292–2215) encompassing these peptide sequences was cloned froma rat brain library using PCR. The cDNA was subcloned into the pGemexexpression vector, expressed in bacteria, and the purified recombinantpolypeptide was immobilized and used in the affinity purification of thepeptide antisera, as described above. Preparation of affinity-purifiedantibodies against brain spectrin has been described previously (Davisand Bennett, 1983). Antibodies against the MAG were purchased fromBoehringer Mannheim (Indianapolis, IN).

Gel electrophoresis and immunoblot analysis. Gel samples of adult ratbrain membranes were prepared as described previously (Kordeli et al.,1995) and fractionated on 3.5–17% exponential gradient gels beforetransfer to nitrocellulose (Davis and Bennett, 1983). Bound antibodieswere visualized using 125I-labeled protein A and autoradiography (Davisand Bennett, 1983).

Immunocytochemical procedures. Immunocytochemistry of frozen sec-tions from rodent sciatic nerves was performed essentially as described(Kordeli et al., 1990). Animals of various ages were killed, and the sciaticnerves were removed by dissection. Sciatic nerves were immediately fixedin 2% paraformaldehyde either overnight at 4°C or for 3 hr at 4°C (forexperiments involving antibodies against the voltage-dependent sodium

channel) before cryopreservation in sucrose and freezing in liquidnitrogen-cooled isopentane. Primary antibodies bound on 4 mm cryosec-tions were visualized with rhodamine-labeled goat anti-rabbit Ig (Cap-pel, West Chester, PA) alone or in combination with fluorescein-labeledmouse monoclonal anti-chicken Ig (clone CH31, Sigma, St. Louis, MO)in double-labeling experiments. Confocal images were collected on aZeiss LSM 410 confocal microscope, and final figures were preparedusing Adobe Photoshop.

RESULTSCharacterization of antibodies against ankyrinG 480/270 kDa, the voltage-dependent sodium channel,neurofascin, and NrCAMFigure 1 shows an immunoblot analysis of total adult rat brainmembranes (lane 19) probed with antibodies raised against anky-rinG 480/270 kDa and the following ankyrin-binding proteins:voltage-dependent sodium channel (lane 2), neurofascin (lane 3),and NrCAM (lane 4). Chicken antibodies against the common“tail” domain of ankyrinG 480/270 kDa recognize two polypep-tides of 480 and 270 kDa as predicted. AnkyrinG 270 kDa arisesfrom ankyrinG 480 kDa by alternative mRNA processing betweennucleotides 7202 and 12446 of the ankyrinG cDNA publishedsequence, removing 1748 amino acids from the ankyrinG “tail”domain (our unpublished data). Antibodies raised against thedeleted sequence specific to ankyrinG 480 kDa recognize nodesof Ranvier in adult sciatic nerve (Zhang and Bennett, 1996) aswell as nodal intermediates in myelinating axons (not shown).Expression of ankyrinG 480 kDa precedes that of ankyrinG 270kDa, which is expressed only after day 10 in the developingnervous system. Antibodies raised against the voltage-dependent

Figure 1. Immunoblot analysis of adult total rat brain membranes withantibodies against ( 1) ankyrinG 480/270 kDa, ( 2) voltage-dependentsodium channel, (3) neurofascin, and (4) NrCAM. Lane 19 shows thebrain membrane preparation stained with Coomassie blue.

7026 J. Neurosci., September 15, 1997, 17(18):7025–7036 Lambert et al. • Ankyrin and the Development of the Node of Ranvier

sodium channel show a single band representing the 260 kDa asubunit, which migrates at 220 kDa in a continuous electro-phoretic buffer system (Srinivasan et al., 1988). These antibodiesalso strongly label nodes of Ranvier in adult sciatic nerve (Daviset al., 1996), confirming earlier antibody localization of thevoltage-dependent sodium channel to the node (Ellisman andLevinson, 1982). Antibodies against FNIII domains 1–4 of neu-rofascin and NrCAM show three polypeptides of 186, 155, and140 kDa for neurofascin as described (Davis et al., 1996) and asingle polypeptide of 150 kDa for NrCAM. The three neurofascinpolypeptides are alternatively spliced products of the rat neuro-fascin gene (Davis et al., 1996), whereas the 150 kDa NrCAMpolypeptide represents the extracellular domain of NrCAM(Kayyem et al., 1992; Davis and Bennett, 1994).

AnkyrinG 480/270 kDa defines a specialized domainwithin the spectrin–actin network at nodal axonsegments of adult nodes of RanvierLocalization of ankyrinG with respect to spectrin was examinedin the adult sciatic nerve by confocal microscopy using double-labeling with rabbit antibody against spectrin and a chicken an-tibody against ankyrinG 480/270 kDa (Fig. 2). Spectrin (Fig. 2A)stains continuously along the axon (Trapp et al., 1989), includingthe nodal and paranodal areas, characterized by a reduction inaxonal caliber. Spectrin staining also occurs in the cytoplasmicprocess of the Schwann cell on the outside edge of the myelinsheath. Antibody against ankyrinG 480/270 kDa (Fig. 2B), incontrast, is localized to a 1–2 mm zone comprising the nodalmembrane, in agreement with initial observations of ankyrinG

staining (Kordeli et al., 1995). The ankyrin binding proteinsNrCAM, neurofascin, and the voltage-dependent sodium channelalso are colocalized with ankyrinG 480/270 kDa at the adult nodeof Ranvier (Davis et al., 1996).

AnkyrinG 480/270 kDa and ankyrinB 440 kDa areco-expressed in an abundant, continuous distributionalong axons before myelinationThe localization of ankyrinG 480/270 kDa and ankyrinB 440 kDawere examined in premyelinated axons of the developing ratnervous system. Extensive immunostaining for both ankyrins wasobserved in axons of both the CNS and PNS in the embryonic day

16 rat. Figure 3 shows the colocalization (arrows) of ankyrinG

480/270 kDa (B, D, F) with spectrin (A), NrCAM (C), and theankyrinB 440 kDa molecule (E) in axons and bundles of axonsemanating from dorsal root ganglia. AnkyrinG 480/270 kDa andankyrinB 440 kDa as well as the ankyrin-binding proteins wereabundantly expressed in these axons and localized continuouslyalong their length. Only faint levels of neurofascin immunostain-ing were observed, confirming observations that suggest a pre-dominantly postnatal expression for this protein in mammals(Davis et al., 1993; Mocosco and Sanes, 1995). No staining wasdetectable in these axons using antibodies to the voltage-dependent sodium channel.

These results demonstrate a major difference between thedistribution of axonal ankyrins in early development and in adultnerve axons. AnkyrinB 440 kDa, which apparently is present insome axons before myelination, disappears from myelinated ax-ons and is present in unmyelinated axons in the adult CNS(Kunimoto et al., 1991; Chan et al., 1993). AnkyrinG 480/270 kDais co-expressed with ankyrinB 440 kDa in embryonic nerve, but inadults it is absent from unmyelinated axons as well as regions ofmyelinated axons in contact with myelin (Kordeli et al., 1995)(Fig. 2).

AnkyrinG 480/270 kDa redistributes to clusterscontaining Na channel, neurofascin, and NrCAMlocalized at sites adjacent to MAG-stainingprocesses of Schwann cells during myelination ofperipheral nerveThe transition from a continuous distribution of ankyrinG 480/270 kDa in axons to a highly polarized localization at the node ofRanvier with low expression in unmyelinated axons in the adultwas studied in myelinating sciatic nerves during early postnataldevelopment of rats. Myelination in the sciatic nerve occursbetween postnatal days 2 and 13, and it occurs at different ratesand times depending on the axon. This heterogeneity allowsvisualization of axons in various stages of myelination in a singlesection of postnatal tissue, but it also presents a problem indistinguishing early stages in myelination. Expression of theMAG provided a marker for the onset of myelination and forSchwann cell processes that contact axons. MAG is expressed by

Figure 2. Immunofluorescence localization of ankyrinG 480/270 kDa with respect to spectrin at the node of Ranvier. A 4 mm cryosection of an adultrat sciatic nerve was double-labeled with an antibody against spectrin ( A) and an antibody against ankyrinG 480/270 kDa ( B). The composite image ( C)was collected by confocal microscopy.

Lambert et al. • Ankyrin and the Development of the Node of Ranvier J. Neurosci., September 15, 1997, 17(18):7025–7036 7027

Schwann cells at ;1.5 turns of the Schwann cell around the axonand is initially localized to the periaxonal interface (Martini andShachner, 1986). As myelination progresses, MAG becomes in-creasingly localized to areas of uncompacted myelin such as theparanodal loops adjacent to the nodal axon segment in adultnodes of Ranvier (Trapp et al., 1989).

Figure 4 shows the localization of ankyrinG 480/270 kDa (A, E)with respect to MAG as a marker for Schwann cell processes (C,D) in the 2-d-old rat sciatic nerve. Although ankyrinG 480/270kDa was still localized predominantly along the length of axons,1- to 2-mm-size clusters of staining (arrowheads) were observed.Clusters were also occasionally observed in pairs ;5–10 mmapart. Ankyrin clusters were localized to gaps between MAGstaining, presumably representing sites of node formation be-tween myelinating Schwann cells. Clusters were also observed atthe ends of MAG-positive fibers. Formation of ankyrinG 480/270kDa clusters correlated with the onset of myelination, as indicated

by the expression of MAG. No ankyrinG 480/270 kDa clusterswere observed in areas of the sciatic nerve with little or noapparent MAG staining.

The localization of ankyrinB 440 kDa with respect to MAGwas also examined in the 2-d-old rat sciatic nerve. AlthoughankyrinB 440 kDa colocalized with ankyrinG 480/270 kDa inpremyelinated axons of the embryonic nervous system (Fig.3E,F), it is expressed at much lower concentrations in myelinat-ing areas (i.e., MAG-positive areas) of the 2-d-old sciatic nerve(Fig. 4B,F). Figure 5 shows the localization of ankyrinB 440 kDa(A) with respect to ankyrinG 480/270 kDa (B) in the 2-d-oldsciatic nerve and indicates that these molecules colocalize inaxons lacking ankyrinG 480/270 kDa clusters. AnkyrinB 440 kDaalso is colocalized with ankyrinG 480/270 kDa in clusters, but it isnot concentrated to the same degree as ankyrinG 480/270 kDa.AnkyrinB 440 kDa also is present in some but not all adult nodesof Ranvier (our unpublished data). The two ankyrins thus may

Figure 3. Colocalization of ankyrinG 480/270 kDa, spectrin, NrCAM, and ankyrinB 440 kDa in dorsal root axons of the embryonic day 16 rat.Cryosections (4 mm) of the dorsal roots from an embryonic day 16 rat were double-labeled with antibodies to ankyrinG 480/270 kDa (B, D, F ) and spectrin(A), NrCAM (C), or ankyrinB 440 kDa (E). Arrows indicate the staining of axons or bundles of axons emanating from the dorsal roots. Insets show asingle bundle of axons at 23 higher magnification.


Figure 4. Localization of ankyrinG 480/270 kDa and ankyrinB 440 kDa with respect to MAG in the 2-d-old rat sciatic nerve. Cryosections (4 mm) ofthe 2-d-old rat sciatic nerve were double-labeled with antibodies to ankyrinG 480/270 kDa (A), ankyrinB 440 kDa (B), and MAG (C, D). Compositeimages collected by confocal microscopy are shown in E and F. Arrows denote the position of ankyrinG 480/270 kDa clusters. Inset shows one of theseclusters at 33 higher magnification.


exhibit some functional overlap. The remainder of this study willfocus on ankyrinG 480/270 kDa.

Colocalization of ankyrinG 480/270 kDa and ankyrin-binding

proteins in developing nerve was assessed at the light level usingdouble-labeling with respect to ankyrinG in the 2-d-old rat sciaticnerve (Fig. 6). AnkyrinG 480/270 kDa (D, E, F) colocalized withthe voltage-dependent sodium channel (G), neurofascin (H), andNrCAM ( I) in paired clusters. In addition, single clusters alsoexhibited this colocalization, and no clusters of ankyrinG 480/270kDa without ankyrin-binding proteins were observed in 94 clus-ters examined. Inference of the colocalization of these moleculesis obviously limited to the resolution of the light microscope inthese experiments and awaits further confirmation by double-labeling immunoelectron microscopy.

The relationship of neurofascin–NrCAM clusters to MAG-staining Schwann cell processes is shown at high magnification inthe 5-d-old rat sciatic nerve (Fig. 7). Neurofascin (Fig. 7A, red) isobserved in paired clusters where MAG-positive fibers ( green)flank both sides of the pair, as well as single clusters associatedwith asymmetrical MAG staining. Overlap in neurofascin andMAG staining is clearly observed (Fig. 7, arrowheads), althoughthe highest density of neurofascin clustering did not overlap withMAG staining and was observed in front of the MAG-positivefiber. Because this antibody also stains paranodal isoforms ofneurofascin in adult nodes of Ranvier (Davis et al., 1996), thisresult suggests that neurofascin might be playing a role in estab-lishment of glial–axonal junctions important to the formationof the paranodal domain. Figure 7B shows the localization ofNrCAM (red) to both paired and single cluster intermediates. Inthe paired intermediate, the unequal accumulation of MAGstaining (Fig. 7B, green) in the developing paranodal area associ-ated with the left-hand cluster suggests differing levels of myeli-nation on either side of the developing node. The NrCAM clus-ters clearly localize outside of MAG staining in front of thedeveloping paranodal area, with no apparent contact with the

Figure 5. Colocalization of ankyrinG 480/270 kDa and ankyrinB 440 kDain the 2-d-old rat sciatic nerve. A 4 mm cryosection of the 2-d-old ratsciatic nerve was labeled with antibodies to ankyrinB 440 kDa (A) andankyrinG 480/270 kDa ( B). The composite confocal image is shown in C.Arrows denote the position of ankyrinG 480/270 kDa clusters.

Figure 6. Co-clustering of ankyrinG and ankyrin-binding proteins inpaired cluster intermediates of the 2-d-old rat sciatic nerve. Cryosections(4 mm) of the 2-d-old rat sciatic nerve were double-labeled with antibodiesagainst ankyrinG (D–F) and the voltage-dependent sodium channel (A),neurofascin ( B), or NrCAM (C). Composite confocal images of individ-ual double-cluster structures are shown in G–I.


myelinating glial cell. A localization pattern similar to that ofNrCAM was observed for ankyrinG and the voltage-dependentsodium channel (data not shown).

Clustering of neurofascin and NrCAM precedesredistribution of ankyrinG 480/270 kDa and thevoltage-dependent Na channelThe previous experiments demonstrate that ankyrinG 480/270kDa and ankyrin-binding proteins concentrate in clusters local-ized adjacent to Schwann cell processes as an initial event inassembly of the node of Ranvier. Double-labeling experimentsrevealed that all clusters that contained ankyrin also had ankyrin-binding proteins (Fig. 6); however, these results do not answer thequestion of which protein arrives first at clusters. A carefulexamination of colocalization of neurofascin and NrCAM withrespect to ankyrinG 480/270 kDa in the day 2 sciatic nerverevealed the presence of neurofascin and NrCAM clusters with-out concomitant clustering of ankyrinG 480/270 kDa (Fig. 8). Incontrast, no examples were found of clustered voltage-dependentsodium channel in the absence of ankyrin (not shown). Figure 8shows rhodamine-labeled clusters (arrows) of neurofascin (A)and NrCAM (B) alone and double-labeled clusters of thesemolecules with ankyrinG 480/270 kDa (arrowheads). These data

suggest that clustering of the cell adhesion molecules neurofascinand NrCAM precedes that of ankyrinG 480/270 kDa and thevoltage-dependent sodium channel in the myelinating sciaticnerve.

The possibility that NrCAM and neurofascin cluster beforeankyrinG 480/270 kDa is further supported by comparison ofthe distribution of these molecules in the 2-d-old sciatic nerve(Fig. 9). AnkyrinG 480/270 kDa is distributed continuouslylocalized along the length of the axon with a number offocused clusters (Fig. 9, arrows) of the molecule ( A). In con-trast, neurofascin (Fig. 9B) and NrCAM ( C) are already highlyclustered (arrows), with very little staining observed along therest of the axon. Double-labeling of these sections with anti-

Figure 7. Immunolocalization of the ankyrin-binding cell adhesion mol-ecules neurofascin and NrCAM with respect to MAG in the 5-d-old ratsciatic nerve. Confocal micrographs show 4 mm cryosections double-labeled with MAG (fluorescein) and neurofascin (rhodamine in A) orNrCAM (rhodamine in B). Arrowheads indicate areas of neurofascin andMAG overlap. Insets show areas of interest at 23 higher magnification.

Figure 8. Colocalization of ankyrinG 480/270 kDa (fluorescein in A andB) with respect to neurofascin (rhodamine in A) and NrCAM (rhodaminein B) in 4 mm cryosections of the 2-d-old rat sciatic nerve. Co-clusters ofankyrinG 480/270 kDa with neurofascin or NrCAM are denoted byarrowheads. Clusters of neurofascin or NrCAM alone are denoted byarrows.


bodies to MAG (Fig. 9D,E,F ) shows that many of these clus-ters are associated with MAG-positive Schwann cells (arrows).In addition, a number of neurofascin (Fig. 9E) and NrCAM ( F)clusters (arrowheads) do not appear to be associated withMAG-positive Schwann cells. Similar clusters were not seenfor ankyrinG 480/270 kDa. These observations suggest that thecell adhesion molecules neurofascin and NrCAM are the pio-neer molecules in the assembly of clusters that subsequentlycontain ankyrin and the voltage-dependent sodium channel.

Formation of adult nodes of Ranvier occurs by fusionof paired ankyrinG–sodium channel–adhesionmolecule clustersThe presence of ankyrin and ankyrin-binding proteins in clus-ters early in myelination suggests that these clusters representthe initial intermediates in the assembly of a node of Ranvier.The relationship between single clusters, paired clusters, andfinally mature nodes of Ranvier was analyzed by measurementsof numbers of single and paired clusters and distances betweenpaired clusters at different stages of development (Table 1).Single clusters are the predominant form at day 2, whereas byday 5 the majority of ankyrinG and ankyrin-binding proteinclusters occur in pairs 5–10 mm apart, and by day 7 very fewsingle clusters are observed. These results suggest that the firststep is formation of single clusters, with a second cluster

subsequently forming at a later time. The asymmetric forma-tion of these cluster pairs may be related to differing levels ofmyelination in the Schwann cells flanking the clusters. Theobservation of differing levels of myelination on either side ofthe developing node has been attributed to the partial auton-omy of adjacent Schwann cells (Allt, 1969).

Measurements of distances between ankyrin clusters at differ-ent stages of development support the idea that pairs of clustersassociated with adjacent Schwann cells flanking the future nodalsite fuse to form the mature node of Ranvier. In the 7-d-old ratsciatic nerve a number of paired intermediates are observed thatare more highly localized and closer together (,5 mm) than thoseseen previously in the 5-d-old rat sciatic nerve. Figure 10 showstwo different 4 mm cryosections of the 7-d-old rat sciatic nervelabeled with antibodies to ankyrinG 480/270 kDa. A number ofdifferent nodal intermediates are visible along with a number ofmature nodes. These different structures were also stained withantibodies to NrCAM, neurofascin, and the voltage-dependentsodium channel (data not shown). Some of the intermediates inFigure 10 (arrows) have been numbered to reflect inferred stagesin the formation of the node of Ranvier. Individual intermediatesfrom each potential stage are also shown at higher magnification.These intermediates also clearly show significant staining of anky-rinG 480/270 in the cytoplasm during early stages of nodal for-

Figure 9. Distribution of ankyrinG 480/270 kDa (A, D), neurofascin (B, E), and NrCAM (C, F ) in 4 mm cryosections of the 2-d-old sciatic nerve. Alsoshown is the localization of these proteins with respect to MAG in the same sections (D–F). Arrows denote clusters of proteins associated withMAG-positive processes; arrowheads show clusters of proteins not associated with MAG-positive processes.


mation and a predominant membrane localization for this mole-cule during later stages.

Formation of nodal intermediates does not requireformation of compact myelinPeripheral myelin protein 22 (PMP-22) is expressed by myeli-nating Schwann cells and incorporated into compacted regionsof peripheral myelin. Mutations in this gene account for thetrembler phenotype in mice, characterized by peripheral nervehypomyelination and continuous Schwann cell proliferationinto adulthood (Henry and Sidman, 1988). In humans, PMP-22mutations are responsible for Charcot-Marie-Tooth (type 1A)syndrome (Valentijn et al., 1992), which is characterized bydecreased peripheral nerve conduction velocities, segmentaldemyelination, and partial remyelination, along with Schwanncell proliferation. To study how the inability to form compactmyelin affects formation of the node of Ranvier, sciatic nervesof 20-d-old trembler mice were stained with antibodies againstankyrinG , neurofascin, and the voltage-dependent sodium

channel. Figure 11 A–C shows the immunolocalization of theseproteins in the sciatic nerves of wild-type (11/11) tremblerlittermates. Interference contrast microscopy (DIC) showsthe presence of compact myelin (Fig. 11 A9), with maturenodes of Ranvier evident as gaps in the myelin sheath (arrows).These nodes clearly label with ankyrinG antibodies (arrows) inthe corresponding fluorescent micrograph (A). Wild-type ma-ture nodes also label with antibodies to neurofascin (Fig. 11 B)and the voltage-dependent sodium channel ( C). Only maturenodes were observed. In contrast, DIC micrographs of thetrembler sciatic nerve (11/tr) show hypomyelination (Fig.11 D9) with an absence of clearly discernible nodal structures.Staining of these sciatic nerves with ankyrinG antibodies, how-ever, shows localization of ankyrinG to structures resemblingmature nodes or potentially single-cluster intermediates (Fig.11 D, arrows) and to structures resembling the paired-clusterintermediates seen in developing sciatic nerves (large arrow-heads). A similar staining pattern was also observed with

Figure 10. Immunolocalization of ankyrinG 480/270 kDa to nodal intermediates in the 7-d-old rat sciatic nerve. Cryosections (4 mm) of the 7-d-old ratsciatic nerve were labeled with antibodies to ankyrinG 480/270 kDa. Arrows indicate nodal intermediates and are numbered to indicate progressive stagesin node formation. Individual nodes from each potential intermediate stage are shown below at higher magnification.

Table 1. Tabulation of nodal intermediates stained by ankyrinG at different times during myelination ofthe rat sciatic nerve

Postnatalday

Number ofnodes ob-served

Relative %of singleclusters

Relative %of pairedclusters(5–10 mm)

Relative %of pairedclusters(,5 mm)

Relative %of maturenodes

2 94 71 15 9 55 74 30 34 20 167 56 9 14 35 42

Cryosections (4 mm) were prepared from the same areas of three separate sciatic nerves, at each time point, and double-stainedfor ankyrinG and MAG. No significant variation in staining was noted between different nerves from the same time point.Single clusters were distinguished from mature nodes by the absence of MAG staining on one side of the cluster.


antibodies to neurofascin (Fig. 11 E) and the voltage-dependent sodium channel ( F).

DISCUSSIONAnkyrinG 480/270 kDa and the three ankyrin-binding integralmembrane proteins neurofascin, NrCAM, and the voltage-dependent sodium channel colocalize at the axonal membrane ofthe adult node of Ranvier in a specialized membrane domainwithin the spectrin–actin network (Kordeli et al., 1995; Davis etal., 1996) (Fig. 2). Before myelination, ankyrinG 480/270 kDa andthe related ankyrin isoform ankyrinB 440 kDa are co-expressedalong with NrCAM in an abundant, continuous distribution alongthe length of axons, whereas neurofascin and the voltage-dependent sodium channel are present at barely detectable levels(Figs. 3, 4). This study has resolved morphological intermediatesin the developmental transition from a continuous distribution ofankyrinG 480/270 kDa in all axons to a highly polarized localiza-tion at the node of Ranvier. The first detected event is formationof clusters containing the cell adhesion molecules neurofascin andNrCAM at sites independent of MAG-staining Schwann cellprocesses. Subsequent steps involve recruitment of ankyrinG 480/270 kDa and the voltage-dependent sodium channel to clustersites defined by the cell adhesion molecules, and elaboration ofMAG-staining Schwann cell processes adjacent to these clustersites. Single clusters with associated Schwann cell processes thenare joined by another cluster–MAG-staining Schwann cell pro-cess to form a pair, and pairs of clusters then fuse to form themature node of Ranvier. In addition to rearrangements ofankyrins, differences in expression of ankyrins also occur duringmyelination: ankyrinB 440 kDa is downregulated from myelinat-ing axons soon after expression of MAG and is retained inunmyelinated axons, whereas ankyrinG 480/270 kDa disappearsfrom unmyelinated axons.

AnkyrinG is a candidate to couple cell adhesion molecules tothe voltage-sensitive sodium channel during assembly of the nodeof Ranvier. This idea is supported by observations that clusters of

neurofascin and NrCAM are joined by ankyrinG 480/270 kDa andthe voltage-dependent sodium channel during differentiation ofmyelinated axons, and biochemical data that suggest a multivalentankyrin membrane-binding domain (Michaely and Bennett,1995a,b). The proposed ankyrinG-mediated link between adhe-sion molecules and ion channels would allow the cell adhesionmolecules to direct the localization of voltage-sensitive sodiumchannels in the axonal membrane. Additional interactions ofadhesion molecules with the voltage-dependent sodium channelalso are likely to occur. For example, the b-2 subunit of thevoltage-dependent sodium channel has homology to the NrCAM-binding protein F11 and potentially could also associate laterallywith NrCAM (Isom et al., 1995).

The observations of this study of early and synchronous clus-tering of ankyrin and the voltage-dependent sodium channelduring differentiation of myelinated axons disagree with the re-port that clustering of the sodium channel precedes that ofankyrin in neuronal–Schwann cell co-cultures (Joe and An-gelides, 1992). This contradiction may result from differences in invivo development versus in vitro cultures. Alternatively, ankyrinantibodies used in the earlier study may not distinguish betweenankyrinG 480/270 kDa and ankyrinB 440 kDa, both of whichcolocalize to premyelinated axons (Fig. 3). The proposal of thisstudy that formation of the node of Ranvier results from the“fusion” of two cluster intermediates composed of ankyrin, thesodium channel, and the cell adhesion molecules is in agreementwith the observations of Shrager and colleagues, who have re-ported sodium channel clusters in single fibers from remyelinating(Dugandzija-Novakovic et al., 1995) and myelinating (Vabnick etal., 1996) sciatic nerve axons.

Clustering of neurofascin and NrCAM could facilitate thesubsequent recruitment and enhance the local concentration ofankyrinG and the sodium channel by several mechanisms. Theankyrin membrane-binding domain contains two binding sites forneurofascin as well as additional sites for the anion exchanger

Figure 11. Immunolocalization of ankyrinG 480/270 kDa, neurofascin, and the voltage-dependent sodium channel in sciatic nerves from thehypomyelinated mutant mouse trembler. Cryosections (4 mm) of sciatic nerves from 20-d-old wild-type (A–C) and trembler (D–F) mice were stained withantibodies against ankyrinG 480/270 kDa (A, D), neurofascin (B, E), and the voltage-dependent sodium channel (C, F ). Nodes of Ranvier are highlightedby arrows. Large arrowheads indicate double-cluster structures in the trembler mutant similar to the nodal intermediates observed in the rat developingsciatic nerve. A9 and D9 represent the corresponding DIC images of the immunofluorescent micrographs shown in A and D and illustrate the lack ofcompact myelin in the trembler mutant. Arrows (A9) indicate the positions of the nodes stained with the ankyrinG antibody in the wild-type sciatic nerve.


(Michaely and Bennett, 1995a,b) and potentially could bind pref-erentially to dimers or higher oligomers of neurofascin. A secondmanner in which the clustering of neurofascin and NrCAM mightaffect binding of ankyrin is through changes in intracellularsignaling pathways. For example, tyrosine phosphorylation ofneurofascin completely abolishes its ability to bind to ankyrin(Garver et al., 1997), and local activation of a tyrosine phos-phatase could lead to recovery of neurofascin–ankyrin binding.In support of these ideas, Dubreuil et al., (1996) demonstratedrecently that concentration of neuroglian, a Drosophila homologof L1, at sites of cell–cell contact is accompanied by recruitmentof Drosophila ankyrin in insect cells.

Recently, Ellisman and colleagues (Deerinck et al., 1997) ob-served that aggregates of ankyrin and the voltage-dependentsodium channel form in regions of nerves of dystrophic mice thatlack Schwann cells, demonstrating that direct Schwann cell–axoncontacts are not required for clusters. These findings contrastwith the results of Shrager and colleagues (Dugandzija-Novakovic et al., 1995; Vabnick et al., 1996), who have demon-strated a role for the Schwann cell in the organization andpositioning of sodium channel clusters. The findings of this studythat clusters of neurofascin and NrCAM precede elaboration ofMAG-staining Schwann cell processes and that ankyrin andsodium channels cluster in the trembler mutant is consistent withthe idea that the axon plays an active role in initial events inmyelination and node formation. A possible explanation for theseconflicting results is that Schwann cells are able to induce clustersof ankyrin and ankyrin-binding proteins in the axonal membrane,which then act as cues for the correct positioning of the Schwanncell during its adherence to the axonal membrane.

Mechanisms for the induction of ankyrin and ankyrin-bindingprotein clusters might include the participation of secreted mol-ecules produced by Schwann cells or possibly other cells. Barresand coworkers (Kaplan et al., 1997) have reported thatoligodendrocyte-conditioned cell-free medium can rapidly initi-ate aggregation of sodium channel and ankyrin in axons ofcultured rat retinal ganglion cells. Interestingly these clustersappear to be spaced at the correct nodal interval, suggesting thatan intrinsic axonal factor might regulate nodal periodicity. Anexample of a potential soluble factor is agrin, which is an extra-cellular matrix protein expressed by Schwann cells as well asother cells, including motor neurons (Ruegg et al., 1992). Agrininduces sodium channel clustering on cultured muscle fibers(Sharp and Caldwell, 1996) and is localized at nodes of Ranvier(Reist et al., 1987).

This study, based on observations in the myelinating sciaticnerve and experiments with the hypomyelinating mutant trembler,presents evidence that clustering of ankyrin and the voltage-dependent sodium channel precedes and does not require theelaboration of compact myelin. Differentiation of the axonalmembrane before the elaboration of compact myelin has beennoted in electron microscopy studies of myelinating nerves (Wax-man and Foster, 1980; Wiley-Livingston and Ellisman, 1980). Thelocalization of ankyrin and the voltage-dependent sodium chan-nel in the trembler mutant contrasts with the localization of theshaker K1 channel, which does not localize to the paranodal areain these mutants (Wang et al., 1995). A potential qualification ininterpreting these results are the cycles of demyelination andremyelination that the trembler mutant exhibits, which may ac-count for the presence of nodal intermediates in the sciatic nervesof these animals. The presence of nodal intermediates in trembler,however, could be relevant to the pathophysiology of human

equivalents of this mutation, such as Charcot-Marie-Tooth (type1A) syndrome (Valentijn et al., 1992). Formation of sodiumchannel clusters could still provide electrophysiological benefitscompared with totally dispersed distribution and could explainthe relatively mild phenotype of this disease. In this case, treat-ments with agents that promote clusters could provide a potentialtherapeutic approach to ameliorate this disease as well as otherdisorders of myelin, such as multiple sclerosis. A candidate forsuch a cluster-promoting factor is the active component ofoligodendrocyte-conditioned medium that induces clustering ofankyrin and sodium channels (Kaplan et al., 1997).

The observation that ankyrinG is confined within a specializedregion of the spectrin–actin network in myelinated axons impliesthat the spectrin-based membrane skeleton in internodal regionsof myelinated axons involves ankyrin-independent attachmentproteins. Spectrin does bind directly to brain membranes lackingankyrin (Steiner and Bennett, 1988), although the identity ofthese sites is not yet known. An ankyrin-based mechanism forpolarity in axons differs from models for formation of basolateraldomains of epithelial cells (Nelson and Veshnock, 1987). In thissystem, the targeted recruitment of both spectrin and ankyrin hasbeen implicated in the polarized distribution of the Na1/K1

ATPase in the basolateral domain.In conclusion, this study has used newly defined molecular

components of the node of Ranvier to discriminate early devel-opmental stages in myelination. The axonal clusters of neurofas-cin and NrCAM described here are the earliest stage in assemblyof the node of Ranvier yet to be resolved. In addition to thesefindings, this work also presents a new set of questions. The basisfor initial clustering of cell adhesion molecules and the role ofextracellular signals versus intrinsic axonal polarity is yet to bedetermined. The role of axonal vesicle transport in the delivery ofankyrinG 480/270 kDa and the voltage-dependent sodium channelto cell adhesion molecule clusters remains to be elucidated. Thenegative signals that exclude MAG-staining Schwann cell pro-cesses from the nodal axon segment also are not understood.Answers to these questions may eventually contribute to detailedunderstanding of the cellular events in myelination and helpdevelop effective strategies to promote remyelination in clinicalsettings.

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